Abstract GPCR signaling pathways regulate many cellular functions including migration. GPCR mediated cell migration is at the basis of immune and tumor cell migration and is central to pathologies such as tumor invasiveness. A number of signaling and cytoskeletal proteins involved in cell migration have been identified. But it is not clear how these molecules respond to extracellular cues and operate together in a spatially dynamic network to govern cell migration. To address this question, in the existing grants, we have focused on the development of optogenetic strategies to control GPCR and G protein subunit signaling. In this proposal for a MIRA, we have expanded the optogenetic strategies to newer targets: to small G proteins -- Cdc42, Rac and RhoA; cytoskeletal proteins; and plasma membrane tension. Our recent results using these strategies show firstly, that selective optical activation of Cdc42 and RhoA can direct the entire range of migratory responses. Secondly, that a novel optogenetic tool that induces localized decrease in plasma membrane tension can lead to cell migration in a direction precisely sensitive to the location of decreased tension. Based on these results, here we propose to examine a model that an extracellular signal evokes spatially coordinated changes in signaling activity, cytoskeletal proteins and localized plasma membrane tension that result in the migratory response. To test this hypothesis we will address the following questions. How does the front of the cell communicate with the back? How does a cell respond globally to localized retraction? What is the function of the uropod? What is the role of adhesion dynamics? How does plasma membrane tension control migration? Optogenetic strategies will be used to perturb signaling activity, cytoskeletal proteins and localized plasma membrane tension. Sensors will be used to detect subcellular molecular and cellular responses. Magnetic tweezers will be used to measure tension across migrating cells. The transition of immune cells such as macrophages and tumor cells from amoeboid to mesenchymal state and back plays an important role in tumor invasiveness. Using transcriptome analysis we will identify genes upregulated in amoeboid cells in comparison to mesenchymal cells and target them optically, genetically and chemically to understand the basis of amoeboid cell movement. We will examine migration in 3D matrices where cells undergo this transition in migratory mode. We anticipate this approach to identify how dynamic interactions between signaling, cytoskeleton and plasma membrane govern migration.